Conjugated polyene sterol derivatives as membrane probes

ABSTRACT

The present invention relates to the synthesis of conjugated polyene sterol derivatives, the compounds obtained and to their use as fluorescent probes for cellular membranes. The fluorescent probes of the present invention resemble cholesterol both structurally and in amphipathic nature. The probes of the present invention have potential for use in determining cholesterol levels and cholesterol properties and cell membrane properties and can be applied to clinical assays and diagnoses involving cholesterol.

This is a division of application Ser. No. 06/867,565, filed May 28,1986, now U.S. Pat. No. 4,879,069.

BACKGROUND OF THE INVENTION AND PRIOR ART

The present invention relates to the synthesis of conjugated polyenesterol derivatives, the compounds obtained and to their use asfluorescent probes for cellular membranes.

A membrane probe is essentially a research tool used to probe(investigate) the environment of a membrane. Membrane probes findapplication, for example, in determining the effect of environmentalpollutants and dietary constituents on cell membranes. Cell membranesare very selective and will permit only selected molecules to permeatethe membrane. As well, the interactions between the membrane and itsenvironment (i.e. the molecules in the environment) are very selective.Membrane probes are useful in the investigation of the structure andfunction of membrane constituents such as proteins and enzymes.Cholesterol is a major membrane constituent. The molecular structure ofthe probes of the present invention is very similar to cholesterol andthus the probes behave in a manner similar to cholesterol.

The probes of the present invention are fluorescent, so their behaviourin the membrane environment and their effect on the membrane can beeasily determined by simply shining light on the membrane and observingthe light emitted by the probe. Because of the very great sensitivity offluorescence techniques, only very small amounts of extrinsic probematerial need be incorporated into the sample of interest. Thiscontrasts with other spectroscopic techniques such as nuclear magneticresonance, absorption and electron spin resonance spectroscopy, whichrequire significantly greater amounts of probe substance, with muchgreater risk of altering the system being investigated. Techniques usingradio-isotopes have a high degree of sensitivity but by their nature aremore hazardous and the radioisotopic probe materials have limited shelflife.

By studying the behaviour of the probe of the invention more can belearned about the behaviour of cholesterol. The probes of the presentinvention have potential for use in determining cholesterol levels andcholesterol properties in membranes and cell membrane properties and canbe applied to clinical assays and diagnoses involving cholesterol.

Aromatic olefins, particularly diphenylhexatriene (DPH), have been usedextensively as fluorescent probes of membrane fluidity, (see forexample, L. A. Chen, R. E. Dale, S. Roth and L. Brand, J. Biol. Chem.,1977, 252, 2163). Such molecules, however, are not natural membraneconstituents and can provide only indirect insight into protein-lipidand lipid-lipid interactions.

Cholesterol is an important lipid component, but its role in influencingmembrane structure and dynamics is poorly understood. Somecholesterol-type molecules in which the C-3 alcohol function has beenmodified have been synthesized. (See for example, R. R. Rando, F. W.Bangerter and M. R. Alecio, Biochim. Biophys. Acta, 1981, 684, 12.) Forexample, the compounds ##STR1##

Apart from a loss of the amphipathic nature of the cholesterol moiety insome of these derivatives (e.g. 1(a)), in all cases the C-3 hydroxyfunction has been drastically altered. Other membrane probes have beensynthesized in which the C-3 hydroxy group is intact. (See for example,R. Bergeron and J. Scott, Anal. Biochem , 1982, 119, 12H; F. Schroeder,FEBS Lett., 1981, 135, 127.) For example, the compounds ##STR2##However, the unsaturation in the ring systems 2(a) and 2(b) drasticallychanges the molecular geometry. Such probes would be expected to packdifferently from cholesterol in membranes and the lipid-probeinteractions would be different from those of lipid-cholesterol.

Fluorescent chromophores in the C-17 side-chain of cholesterol have beenreported (See for example, Y. J. Mao, A. K. Soutar, K.-Y. Hong, H. J.Pownall and L. C. Smith, Biochemistry, 1978, 17, 2689.) For example, thecompounds ##STR3##

In compounds of structures 3(a) a, 3(b) and 3(c) ad the hydrophobiccharacter of the C-17 side-chain is much different from that ofcholesterol. The membrane probes of the present invention, which have anolefinic fluorescent chromophore in the C-17 side-chain, resemblecholesterol more closely both in geometry and in amphipathic nature.

SUMMARY OF THE INVENTION

The present invention is directed to olefinic sterol derivatives of theformula I ##STR4## wherein

R represents H or an acyl group suitable for use in cholesterol esteraseassays, for example, a formyl, C₂ -C₂₀ alkylcarbonyl, C₃ -C₂₀alkenylcarbonyl or C₃ -C₂₀ alkynylcarbonyl group or an arylcarbonylgroup and

A represents ##STR5## in which R¹ represents H, C₁ -C₄ lower alkyl, C₂-C₄ lower alkenyl, C₂ -C₄ lower alkynyl or aryl, preferably phenyl orsubstituted phenyl wherein the substituent is consistent withfluorescence and R² represents --(CH═CH)_(n) --CH═CH₂, --(CH═CH)_(n)-phenyl, --(CH═CH)_(n) -naphthyl, --(CH═CH)_(n) -tricyclic aryl--(CH═CH)_(n) -tetracyclic aryl or ##STR6## in which

is 0 to 3 and R and R¹ are as defined above.

When R¹ is substituted phenyl, the substituents must be consistent withfluorescence. By a substituent which is consistent with fluorescence wemean a substituent which may enhance fluorescence, for example fluorine,chlorine or an aryl group, preferably phenyl, or a substituent whichwill not detract from fluorescence, for example lower alkyl such asmethyl or ethyl.

Tricyclic aryl groups which can be present as part of the R² moietyinclude anthracene and phenanthrene. Tetracyclic aryl groups which canbe present as part of the R² moiety include naphthacene,1,2-benzanthracene, chrysene and pyrene.

Specifically, the compounds of the present invention can be representedby the following structures: ##STR7## wherein

R, R¹, R² and n are as defined above.

Structures of formulae Ia, Ib and Id are preferred. Particularlypreferred are structures of formulae Ia and Ib.

Of particular interest are the compounds obtained when R¹ is methyl andR² is --CH═CH)-phenyl, when R¹ is phenyl and R² is --(CH═CH)-phenyl,when R¹ is methyl and R² is naphthyl and when R¹ methyl and R² isphenyl.

The compounds of the present invention can be prepared by reacting acompound of formula II ##STR8## its isomer of formula II' ##STR9## inwhich

R' is R as defined above or a protecting group and R¹ is as definedabove with a base and a triphenylphosphonium compound of the formula##STR10## or a phosphonate compound of the formula ##STR11## in which

R² is as defined above,

R⁴ is a C₁ -C₄ lower alkyl group and X⁻ is halide or other suitablenucleophilically displaceable group, and if required removing theprotecting group.

It is preferred that R' is a protecting group. The preferred method ofobtaining a compound of formula I in which R is acyl is to use acompound of formula II or II' in which R' is a protecting group, todeprotect to obtain a compound of formula I in which R is hydrogen andthen to acylate to obtain a compound of formula I in which R is an acylgroup. Acylation of cholesterol compounds at the 3-hydroxy position canbe carried out by well known methods and usually proceeds inquantitative yield.

The protecting group must be a base stable group and is preferably asubstituted silyl protecting group. Most preferably the protecting groupis t-butyldimethylsilyl or t-butyl diphenylsilyl. The protecting groupcan be removed, for example, by reaction with tetrabutylammcniumfluoride or acetic acid, water and tetrahydrofuran.

The base can be selected from the group consisting of, for example,n-butyllithium, lithium diisopropylamide, sodium hydride, lithiumhydride, sodium methoxide, sodium ethoxide, sodium isopropoxide andpotassium t-butoxide.

In a preferred feature R⁴ is ethyl and X⁻ is chloride or bromide.

The reaction between the compound of formula II or II' and thetriphenylphosphonium compound can be carried out in tetrahydrofuran(THF) as solvent. When using a compound of formula II, if it is desiredto increase the ratio of compound Ic to Ia in the product, there can beused as solvent a mixture of hexamethylphosphoramide (HMPA) and THF,suitably 10% HMPA and 90% THF. Similarly when using a compound offormula II' the ratio of compound Id to Ib in the product can beincreased by using as solvent a mixture of HMPA and THF.

The compounds of formula II and II' can be prepared from a compound offormula IV ##STR12## in which

R' and R¹ are as defined above. In one embodiment, the compound offormula IV is reacted with diethyl 2-(cyclohexylimino) vinyl phosphonateand the crude product hydrolysed, for example on a silica gel column togive the compounds of formulae II and II'. The diethyl2-(cyclohexylimino)vinyl phosphonate can be obtained by hydrolysis ofdiethylphosphonoacetaldehydediethyl acetal and subsequent condensationwith cyclohexylamine.

In an alternative embodiment the compound of formula IV is reacted withvinyl magnesium bromide with further hydrolysis to obtain a compound offormula III ##STR13## in which

R' and R¹ are as defined above.

The compound of formula III is then oxidatively rearranged.

In a preferred feature the oxidative rearrangement is carried out inmethylene chloride in the presence of pyridinium chlorochromate,imidazole and sodium acetate which induce allylic rearrangement of thehydroxyl group and its oxidation to an aldehyde group.

The oxidative rearrangement can also be achieved by a first allylicrearrangement under mildly acidic conditions followed by oxidation witha mild oxidizing agent, for instance, manganese dioxide, Moffat'sreagent or 2,3-dichloro-5,6-dicyanobenzoquinone.

The isomeric compounds of formula II and II' can be purified andseparated into isomers by chromatography, preferably by columnchromatography or by high performance liquid chromatography.

For a more direct method of obtaining compounds of formula I withconfiguration (b), the compound of formula IV (in which R' and R¹ are asdefined above) is treated with a phosphonate compound of formula##STR14## and a base. Suitable bases include those mentioned above. IfR' is a protecting group, the protecting group must be a base stablegroup and is preferably a substituted silyl protecting group. Mostpreferably the protecting group is t-butyldimethylsilyl ort-butyldiphenylsilyl. The protecting group can be removed, for example,by reaction with tetrabutylammonium fluoride or acetic acid, water, andtetrahydrofuran.

The present invention is also directed to a method of analyzingcholesterol properties and cell membrane properties wherein a compoundof formula I is admixed with a membrane sample and the compound-sampleadduct is subjected to absorption or fluorescence spectroscopy. In oneembodiment, the membrane sample is suspended in an aqueous medium,buffered, if necessary, to a pH between 6 and 8, preferably atphysiological pH, which is 7.4. Examples of preferred buffers includephosphate buffer tris(hydroxymethyl)amino methane hydrochloride (TRIS)and sodium cacodylate. To the suspended sample there is added a verysmall volume of a solution of known concentration of the compound offormula I. Suitable solvents include methanol, ethanol, chloroform,acetone, dimethylsulfoxide and THF. The mixture is vortexed immediately,incubated at a suitable temperature, preferably 37° C., for a timesufficient to incorporate the compound of formula I in the membrane. Thecompound-sample mixture may or may not be subjected to sonication tohelp the incorporation. The supernatant liquid is removed and issubjected to absorption, fluorescence or polarized fluorescencespectroscopy.

In an alternative embodiment, an aqueous suspension of the membranesample is added to a dried film of a compound of formula I on the insideof a round bottomed flask. The obtained mixture is then vortexed andsubjected to the other steps described above to incorporate the compoundof formula I in the membrane. The supernatant is again removed andsubjected to absorption, fluorescence or polarized fluorescencespectroscopy.

In fluorescence polarization spectroscopy, polarized light is directedat the sample under investigation, and measurements of the amount offluorescent light from the sample polarized parallel and perpendicularto the incident polarization direction are taken. Measurement of eitherpolarization, polarization ratio or anistropy can give information aboutthe fluidity of the sample under investigation. For example, if theratio of the intensities of parallel to perpendicular polarized light(polarization ratio), is 1 or close to 1, in the absence of instrumentaland experimental artifacts such as monochromator polarization bias, thenthe probe molecule moves relatively freely in the membrane and themembrane is said to be fluid in the region of the probe. If the ratio isgreater than 1 the movement of the probe molecule is more restricted inthe membrane and the membrane is not as fluid in the region of theprobe. Hence the probes of the invention permit measurements of cellmembrane fluidity and therefore permit studies to determine whatrelationships may exist between fluidity of its membrane and otherproperties of a cell. Other membrane properties can also be determined.As well, because the probe molecule is very much like cholesterol thebehaviour of the probe molecule can be useful in predicting howcholesterol will behave in a similar membrane environment. The probemolecule, which is detectable by virtue of its fluorescence, can be usedas labelled cholesterol. For instance, half of all deaths in the UnitedStates are caused by atherosclerosis, the disease in which cholesterol,accumulating in the wall cf arteries, forms bulky plaques that inhibitthe flow of blood until a clot eventually forms, obstructing an arteryand causing a heart attack or stroke. Labelled cholesterol, in the formof the probe molecules of this invention, can be used in theinvestigation of the process of atherosclerosis and its early diagnosis.

The present invention is also directed to a kit for determiningcholesterol levels and cholesterol properties in membranes and cellmembrane properties, which kit comprises a known concentration of thecompound of formula I, a standard or blank, such as dimyristoylphosphatidylcholine lipid or other lipids for vesicle or liposomepreparation, and, if necessary, buffer solution.

The invention will be further illustrated by reference to the followingExamples.

EXAMPLE 1

The t-butyldimethylsilyl ether (R'=Si(CH₃)₂ C(CH₃)3) of pregnenolone (R¹=CH₃) was treated with vinylmagnesium bromide to give a quantitive yieldof a 90:10 mixture of epimeric C-20 alcohols of formula III (determinedby integration of the 23-H peaks centred at δ5.27 and 5.18; δ5.09) and5.00). Diastereoisomeric aldehydes of formulae II and II' were obtainedin 88% yield by oxidative rearrangement of the epimeric alcohols withpyridinium chlorochromate, imidazole and sodium acetate in CH₂ C₂ atroom temperature for 20 hours. The E- and Z-aldehydes were assigned onthe basis of their 21-CH₃ 300 MHz 'H n.m.r. resonances [in formula II'R¹ =CH₃, δ(21-CH₃) 2.20; in formula II R¹ =CH₃ δ(21-CH₃)1.99]and wereformed in an 80:20 ratio respectively [determined by integration of the23-H peaks at δ10.07 (E) and δ9.97 (Z) and the 22-H peaks at δ6.05 (Z)and δ5.94 (E)]. Separation of the isomers was effected by reversed phasehigh performance liquid chromatography (Altex Ultraspher™ ODS 10 mm×25cm column, 100% CH₃ CN). The overall yield of 70% for the E-isomer offormula II' is an improvement over the methods used previously for theanalogous C-3 acetoxy E-isomer. (See Y. Letourneaux, M. M. L. Lo, N.Chaudhuri and M. Gut. J. Org. Chem. 1975, 40, 516 and A. O. Colonna andE. G. Gros, J. Steroid Biochem., 1973, 4, 171).

The E-aldehyde (in formula II' R¹ ═CH₃), was treated withE-cinnamyltriphenylphosphonium chloride and n-butyllithium intetrahydrofuran giving a 75% yield of a mixture of conjugated trieneswhich were deprotected at C-3 with tetrabutylammonium fluoride. From 'Hn.m.r. analysis ('H- 'H correlated spectrum or COSY, coupling constants,integration and computer simulation in the olefinic proton region) themixture of isomeric alcohols was assigned to a 39:61 ratio of the EEE[in formula Ib R═H, R.sup.═CH₃, R² ═(CH═CH)-phenyl] and the EZE [informula Id R=H, R¹ =CH₃, R² ═(CH═CH)-phenyl] trienes. The integrationexperiments focused on the 25-H signal of the EEE-isomer centred atδ6.86 and the 6-H signal from both isomers at δ5.34. Separation of theseisomers was achieved by reversed-phase high performance liquidchromatography (Altex Ultrasphere™ ODS 10 mm×25 cm column, 98:2 CH₃CN:H₂). The characteristic n.m.r. signals for the olefinic protons inthe side-chain of each of the isomers is given in Table 1.

                  TABLE 1                                                         ______________________________________                                        Chemical shifts and coupling constants of olefinic pro-                       tons in the isomeric conjugated trienes [in formula Ib R = H,                 R.sup.1 = CH.sub.3, R.sup.2 = (CH═CH)-phenyl and in formula Id R =        H,                                                                            R.sup.1 = CH.sub.3, R.sup.2 -- = (CH═CH)-phenyl].                                                 δ(J in Hz)                                            EEE isomer        EZE isomer                                                  (in formula Ib R = H,                                                                           (in formula Id R = H,                                       R.sup.1 = CH.sub.3, R.sup.2 =                                                                   R.sup.1 = CH.sub.3, R.sup.2 =                         Proton                                                                              --(CH═CH)-phenyl)                                                                           --(CH═ CH)-phenyl)                                ______________________________________                                        22    5.98 (11.3)       6.45 (12.3)                                           23    6.62 (11.3, 14.5) 6.32 (11.2, 11.2)                                     24    6.30 (14.6, 10.8) 6.08 (11.1, 11.1)                                     25    6.86 (10.8, 15.6) 7.2-7.3 (under                                                                aromatic proton)                                      26    6.49 (15.5)       6.54 (15.4)                                           ______________________________________                                    

The EEE-isomer isolated showed no detectable impurities and exhibitedm.p. 171.5°-173° C.; λ_(max) ^(abs) 331 nm; 42000 dm³ cm⁻¹ mol⁻¹(methylcyclohexnae); λ_(max) ^(fl) 390 nm (λ^(ex), 330 nm,methylcyclohexane).

EXAMPLE 2

The procedure of example 1 was followed, except that the E aldehyde [informula II', R'═Si(CH₃)_(b) 2 C(CH₃)₃, R¹ ═CH₃ ] was treated withdiethyl cinnamyl phosphonate and lithium diisopropylamide intetrahydrofuran for 24 hours (-78° C.→ room temperature). The resultingmixture of conjugated trienes was deprotected at C-3 withtetrabutylammonium fluoride. The EEE and EZE triene [Ib and Idrespectively with R═H, R¹ ═CH₃, R² ═-CH═CH-phenyl] ratio was determinedby high performance liquid chromatography to be 83:17 (Varian™SPC-18column, 4 mm×15 cm, 2% H₂ O, 98% CH₃ CN, 330 nm). This procedure is thusmore selective than the procedure of example 1 for synthesis of the EEEtriene.

The EEE & EZE trienes were separated by high performance liquidchromatography (Beckman Ultraspher™ ODS column 10 mm×25 cm, 98:2 CH₃ CN:water, 330 nm). The EEE triene was dissolved in pyridine and heated (60°C.) with acetic anhydride for one hour to yield EEE triene 3β-acetate(in formula Ib R═CH₃ CO, R¹ ═CH₃, R² =--CH═CH-phenyl). The crude productobtained was a yellow oily liquid. Identification was made by massspectrometry; m/z=484 g/mole (M⁺), m/z=424 g/mole (M⁺ --CH₃ CO₂ H).

EXAMPLE 3

The procedure of example 1 was followed, except that the E aldehyde [informula II', R'═2 Si(CH₃)₂ C(CH₃)₃, R¹ ═CH₃ ] was treated with cinnamyltriphenylphosphonium chloride and n-butyllithium in tetrahydrofurancontaining 10% of hexamethylphosphoramide for 24 hours (-78° C.→ roomtemperature). The resulting mixture of trienes was deprotected at C-3using tetrabutylammonium fluoride. The EEE and EZE triene [Ib and Idrespectively with R═H, R¹ ═CH₃, R² ═-CH═CH-phenyl] ratio was determinedby high performance liquid chromatography to be 1:99 (Varian™ SPC-18column, 4 mm×15 cm, 2% H₂ 98% CH₃ CN, 330 nm). This method is the one ofchoice for synthesis of the EZE triene.

EXAMPLE 4

Compound Ib[R═H, R¹ ═CH₃, R² ═--(CH═CH)_(n) -phenyl where n═0] andId[R═H, R¹ ═CH₃, R² ═--(CH═CH)_(n) -phenyl where n═0] been synthesized.The aldehyde II' [R'═Si(CH₃)₂ C(CH₃)₃, R¹ ═CH₃ ]was treated withbenzyltriphenylphosphonium bromide and n-butyllithium intetrahydrofuran. The resulting 60/40 mixture of EZ/EE isomers wasdeprotected at C-3 with tetrabutylammonium fluoride in tetrahydrofuran.Identification of the mixture was made by H'n.m.r. analysis and thechemical shifts are indicated in the Table 2 below.

                  TABLE 2                                                         ______________________________________                                        Chemical shifts of olefinic protons in the isomeric                           conjugated dienes [in formula Ib R = H, R.sup.1 = CH.sub.3,                   R.sup.2 = (CH═CH).sub.n -phenyl                                           where n = 0 and in formula Id R = H,                                          R.sup.1 = CH.sub.3, R.sup.2 = (CH═CH).sub.n -phenyl where n = 0].                                   EZ isomer δ                                             EE isomer δ                                                                             (Id, R = H,                                                   (Ib, R = H, R.sup.1 = CH.sub.3,                                                               R.sup.1 = CH.sub.3,                                 Proton    R.sup.2 = phenyl)                                                                             R.sub.2 = phenyl)                                   ______________________________________                                        22        6.36            6.09                                                23        7.11            7.22                                                24        6.47            6.53                                                ______________________________________                                    

The mixture of dienes Ib and Id has an absorption maximum at 290 nm anda fluorescence maximum at 350 nm in methylcyclohexane.

EXAMPLE 5

The t-butyldimethyl silyl ether of pregnenolone (in formula (IV),R'═Si(CH₃)₂ C(CH₃)₃, R¹ ═CH₃) was treated with diethyl cinnamylphosphonate and n-BuLi in tetrahydrofuran for 24 hours (-78° C.→ roomtemperature). Workup of the reaction product yielded a substance whichshowed one fluorescent spot (R_(f) ═0.85,19:1 hexane:ethyl acetate) bythin layer chromatography and a retention time of 50 minutes by highperformance liquid chromatography (Beckman Ultraspher™ ODS, 10 mm×25 cm,95% Methanol, 5% Hexane, 300 nm, 3 ml/min). The fluorescence spectrum ofthe HPLC purified product was measured in 19:1 methanol:hexane and foundto have a fluorescence maximum at 350 nm and to be identical with thatof the product of Example 4. The crude product formed was alsodetermined to contain EE diene [in formula Ib,

R'═Si(CH₃)2C(CH₃)₃, R¹ ═CH₃, R² ═(CH═CH)n-phenyl (where n═0)] bycomparison of its 'H NMR spectrum with the data given in Table 2.

EXAMPLE 6

Diethylphosphonoacetaldehyde diethyl acetal was heated with an 8%solution of hydrochloric acid and a few crystals of hydroquinone at 75°C. for 16 hours. The reaction mixture was distilled to give pureformylmethylphosphonate (70°-72° C., 0.33 mm) which was then condensedwith cyclohexylamine in methanol at 0° C. for 3 hours to give diethyl2-(cyclohexylimino) vinyl phosphonate. The t-butyldimethylsilyl ether ofpregnenolone [in formula IV, R'═Si(CH₃)₂ C(CH₃)₃, R¹ 50 CH₃ ] wastreated with the above vinyl phosphonate and sodium hydride intetrahydrofuran initially at 0° C. and then at reflux for 12 hours. Theresulting reaction mixture was chromatographed on silica gel withhexane/ethyl acetate to give a mixture of the E-aldehyde [in formulaII', R'═Si(CH₃)₂ C(CH₃)₃, R¹ ═CH₃ ] and the Z-aldehyde [in formula II,R'═Si(CH₃)₂ C(CH₃)₃, R¹ ═CH₃ ] . The ratio of aldehydes was determinedby high performance liquid chromatography to be E:Z as 96:4 (Varian™SPC-18 column, 4 mm×15 cm, 100% CH₃ CN, 250 nm).

The E-aldehyde was separated from its Z isomer by high performanceliquid chromatography (Serva™ODS 100 Polyol, 5 m, 22 mm×50 cm, 100%MeOH, 254 nm) and then treated with diethyl 2-methylnaphthyl phosphonateand n-butyllithium in tetrahydrofuran for 24 hours (-78° C.→ roomtemperature) to give a 55% yield of a mixture of the EE and EZ naphthyldienes [Ib and Id respectively, with R═Si(CH₃)₂ C(CH₃)₃, R¹ ═CH₃ , R²=naphthyl]. This mixture was deprotected at C-3 with tetrabutylammoniumfluoride.

The identity of the deprotected compounds was confirmed by massspectroscopy, with an m/z value of 466 g mol⁻¹.

The ratio of EE to EZ naphthyldienes was determined by reversed phasehigh performance liquid chromatography (Beckman Ultraspher™ ODS Column,10 mm×25 cm, 95% methanol, 5% water, λ═340 nm) to be 99:1. This ratiooverstates the amount of EE isomer due to its higher extinctioncoefficient, i.e. greater absorption, at 340 nm. It is estimated thatthe ratio of EE:EZ isomers is at least 95:5.

Fluorescence Studies of Lipid Vesicles

The excited singlet state decay time (fluorescence lifetime) of the EEEtriene Ib[R═H, R¹ ═CH³, R² ═--CH═CH-phenyl] was determined to be 30+5 psin free solution (tetrahydrofuran). Lifetime measurements at 20° C. ofthe EEE triene probe when incorporated in vesicles of the lipiddimyristoyl phosphatidyl choline (DMPC) [200μ1(2.5×10⁻⁵ M probe in THF).250 μI(35 mg/ml of DMPC in CH₃ Cl), 1.55 ml(20 mM tris-acetate buffer,pH 7.2); ratio of lipid to probe approximately 200:1] are shown in Table3 below.

                  TABLE 3                                                         ______________________________________                                        Fluorescence lifetimes and fractional fluorescence of                         the EEE triene in DMPC vesicles.                                                               Lifetime, τ                                              Percent Cholesterol                                                                            Fraction, F                                                  ______________________________________                                         0%              τ.sub.1 = 0.93 ns, τ.sub.2 = 0.26 ns                                  F.sub.1 = 0.66, F.sub.2 = 0.34                               10%              τ.sub.1 = 1.22 ns, τ.sub.2 = 0.31 ns                                  F.sub.1 = 0.47, F.sub.2 = 0.53                               ______________________________________                                    

Steady state fluorescence polarization and anisotropy measurements weremade on the above vesicles in the gel phase (15° C.) and the liquidcrystalline phase (30° C.). Results are given in Table 4.

                  TABLE 4                                                         ______________________________________                                        Steady state fluorescence and anisotropy values of the                        EEE triene in DMPC vesicles.                                                                             30° C. (Liquid                              Percent Cholesterol                                                                         15° C. (Gel Phase)                                                                  Crystalline Phase)                                 ______________________________________                                         0%           (a)*2.03     (a) 2.01                                                         (b) 0.34     (b) 0.34                                                         (c) 0.255    (c) 0.252                                          10%           (a) 1.54     (a) 1.69                                                         (b) 0.21     (b) 0.26                                                         (c) 0.15     (c) 0.19                                           ______________________________________                                         *(a)=Polarization Ratio, (b)=Polarization, (c)=Anistropy                 

Fluorescence Studies of Oriented Lipid Samples

A methanol solution (0.1 mM,600/μ1) of the EEE triene Ib [R═H, R¹ ═CH₃,R² ═-CH═CH-phenyl] was mixed with egg phosphatidylcholine (15 mg; lipidto probe ratio approximately 250:1). After drying under nitrogen andthen under vacuum for 24 hours, the sample was left in a dessicator inan atmosphere of water vapour (saturated solution of K₂ SO₄ in water)for 12 hours at room temperature.

The resulting lipid-water mixture was applied to two microscope coverglasses which were slid over each other to assist in orienting thesample. The orientation was checked by looking for a uniform colourunder a polarization microscope. After another 12 hours in the watervapour atmosphere and another check of the orientation, the two coverglasses were sealed with a two-Component glue and blackened around theedges.

The same procedure was followed for a second sample except that 20% ofthe lipid component was cholesterol.

Steady-state fluorescence polarization measurements (λex═330 nm,)λem═380 nm, photon counting detection) of the two samples were made,varying the angles α and β (see accompanying drawing):

α=0°, 10°, 20°. . . 60°β═130°, 140°, 150°. . . 220° with ρβ160°.

The results are shown in Tables 5 and 6.

                  TABLE 5                                                         ______________________________________                                        Steady-state fluorescence polarization of the EEE triene                      in oriented samples of egg phosphatidyl choline.                                       Polarization           Polarization                                  α(°)β(°)                                                      Ratio (±0.01)                                                                            α(°)β(°)                                                      Ratio (±0.01)                              ______________________________________                                        0 130    2.04          40 130   1.48                                          140      2.19          140      1.56                                          150      2.33          150      1.68                                          160      2.45          160      1.82                                                                 170      1.95                                          10 130   1.87          180      2.06                                          140      1.96          190      2.16                                          150      2.11          200      2.26                                          160      2.26                                                                 170      2.38          50 130   1.38                                                                 140      1.46                                          20 130   1.72          150      1.57                                          140      1.82          160      1.70                                          150      1.92          170      1.87                                          160      2.07          180      2.08                                          170      2.19          190      2.15                                          180      2.33          200      2.21                                                                 210      2.27                                          30 130   1.59                                                                 140      1.68          60 130   1.33                                          150      1.80          140      1.41                                          160      1.93          150      1.49                                          170      2.04          160      1.63                                          180      2.17          170      1.81                                          190      2.28          180      2.02                                                                 190      2.27                                                                 200      2.27                                                                 210      2.26                                                                 220      2.30                                          ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Steady-state fluorescence polarization                                        of EEE triene in oriented samples of                                          80% egg phosphatidyl choline, 20% cholesterol.                                         Polarization           Polarization                                  α(°)β(°)                                                      Ratio (±0.005)                                                                           α(°)β(°)                                                      Ratio (±0.005)                             ______________________________________                                        0 130    2.166         40 130   1.519                                         140      2.214         140      1.610                                         150      2.276         150      1.729                                         160      2.282         160      1.866                                                                170      2.008                                         10 130   1.983         180      2.149                                         140      2.059         190      2.267                                         150      2.101         200      2.416                                         160      2.225                                                                170      2.469         50 130   1.462                                                                140      1.524                                         20 130   1.777         150      1.642                                         140      1.877         160      1.770                                         150      1.998         170      1.923                                         160      2.111         180      2.068                                         170      2.227         190      2.211                                         180      2.466         200      2.330                                                                210      2.452                                         30 130   1.635                                                                140      1.725         60 130   1.405                                         150      1.843         140      1.485                                         160      1.979         150      1.596                                         170      2.108         160      1.729                                         180      2.234         170      1.859                                         190      2.379         180      2.003                                                                190      2.144                                                                200      2.270                                                                210      2.403                                                                220      2.528                                         ______________________________________                                    

Based on the assumption that the fluorescent side chain of the probe hascylindrical symmetry the data were analysed (C. Zannoni, A. Arcione andP. Cavatorta, Chem. Phys. Lipids, 1983 32 179) to give order parameters(maximum possible range 0 (low order)→1 (high order)) as shown below:

    ______________________________________                                        % Cholesterol                                                                 in sample     Order Parameter, S                                              ______________________________________                                         0            0.1                                                             20            0.9                                                             ______________________________________                                    

These results confirm that the probe reports on the fluidity and orderof lipid systems. Further they suggest that the compound of theinvention associates with the cholesterol domains in the sample. Hencethe invention provides probes which can be used as "labelledcholesterol" in an environment with cholesterol molecules and that the"labelled cholesterol" will behave in a similar manner to cholesterol.The behaviour of cholesterol molecules in situ in membranes and theireffect on the lipid order can be observed.

what we claim as our invention is:
 1. A method of analyzing cholesterollevels or properties of cholesterol, said method comprising admixing acholesterol-containing sample with an olefinic compound of formula I##STR15## wherein R represents H, formyl C₂ -C₂₀ alkylcarbonyl, C₃ -C₂₀alkenylcarbonyl, C₃ -C₂₀ alkynylcarbonyl or arylcarbonyl, and Arepresents ##STR16## in which R¹ represents H, C₁ -C₄ lower alkyl, C₂-C₄ lower alkenyl, C₂ -C₄ lower alkynyl, phenyl or phenyl substituted bya substituent selected from the group consisting of halo, aryl and loweralkyl, andR₂ represents --(CH═CH)_(n) --CH═CH₂ _(n) -phenyl,--(CH═CH)_(n) -naphthyl, --(CH═CH)_(n) -tricyclic aryl, ---(CH═CH)_(n)-tetracyclic aryl or ##STR17## in which n is 0 to 3 and R and R¹ are asdefined above, and subjecting the produced compound-sample mixture toabsorption, fluorescence or polarized fluorescence spectroscopy as ameasure of the cholesterol present in the sample.
 2. A method ofanalyzing cholesterol levels or properties in membrane or lipid systemsand cell membrane properties, said method comprising admixing a membranesample with an olefinic compound of formula I ##STR18## wherein Rrepresents H, formyl, C₂ -C₂₀ alkylcarbonyl, C₃ -C₂₀ alkenylcarbonyl, C₃-C₂₀ alkenylcarbonyl or aryl carbonyl, and A represents ##STR19## inwhich R¹ represents H, C₁ -C₄ lower alkyl, C₂ -C₄ lower alkenyl, C₂ -C₄lower alkynyl, phenyl or phenyl substituted by a substituent selectedfrom the group consisting of halo, aryl and lower alkyl, andR²represents --(CH═CH)_(n) --CH═CH₂, --CH═CH)_(n) -phenyl, --(CH═CH)_(n)-naphthyl, --(CH═CH)_(n) -tricyclic aryl, --(CH=CH)n -tetracyclic arylor ##STR20## wherein n is 0 to 3 and R and R¹ are as defined above, andsubjecting the produced compound-sample mixture to absorption,fluorescence or polarized fluorescence spectroscopy as a measure of thecholesterol present in the sample.
 3. A method as claimed in claim 2,comprising the steps of:suspending the membrane sample in an aqueousmedium to produce an aqueous suspension; adding to the aqueoussuspension a known amount of a solution or suspension of the compound offormula I; incubating the produced compound-sample mixture to atemperature about 37° C. for a time sufficient for the compound offormula I to be incorporated in the membrane sample; and subjecting thesupernatant liquid of the mixture to absorption, fluorescence orpolarized fluorescence spectroscopy as a measure of the cholesterolpresent in the sample.
 4. A method according to claim 3 wherein saidaqueous suspension is buffered with a buffer to a pH of about 7.2 andsaid known amount of a solution or suspension of the compound of FormulaI comprises a sonicated aqueous suspension of said compound of FormulaI.
 5. A method according to claim 4 wherein said buffer is a phosphatebuffer.
 6. A method according to claim 3 wherein the supernatant issubjected to fluorescence spectroscopy.
 7. A method according to claim 3wherein the supernatant liquid of the mixture is subjected to polarizedfluorescence spectroscopy.
 8. A method as claimed in claim 3, andfurther including the step of buffering the aqueous suspension with abuffer to a pH between 6 and 8 prior to said incubation.
 9. A kit foranalyzing cholesterol levels or properties in membrane or lipid systemsand cell membrane properties, comprising a known concentration of anolefinic compound of formula I ##STR21## wherein R represents H, formyl,C₂ -C₂₀ alkylcarbonyl, C₃ -C₂₀ alkenylcarbonyl, C₃ -C₂₀ alkynylcarbonylor arylcarbonyl and A represents ##STR22## in which R represent H, C₁-C₄ lower alkyl, C₂ -C₄ lower alkenyl, C₂ -C₄ lower alkynyl, phenyl orphenyl substituted by a substituent selected from the group consistingof halo, aryl and lower alkyl,R² represents --(CH═CH)_(n) -CH═CH₂,--(CH═CH)_(n) -phenyl, --(CH═CH)_(n) -naphthyl, --(CH═CH)_(n) -tricyclicaryl, --(CH═CH)_(n) -tetracyclic aryl or ##STR23## in which n is 0 to 3and R and R¹ are as defined above, and a standard or blank which is purelipid or vesicle or liposome preparation.
 10. A kit according to claim 9wherein said standard is dimyristoyl phosphatidylcholine.
 11. A kitaccording to claim 9 additionally comprising aqueous phosphate buffer.